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Biochemical Defects Associated with Cancer-Causing Pathogenic Mutations in Human MLH1 Andrew Nguyen Laboratory of Dr. Andrew Buermeyer Department of Environmental and Molecular Toxicology Oregon State University Deaths Caused by Cancer – United States, 2005 Men 291,270 deaths Women 273,560 deaths Colon and rectum 10% Approximately 56,000 deaths from colorectal cancer in 2005 alone ©2005 American Cancer Society, National Cancer Institute Why DNA repair is important DNA can be damaged by mutagens (ex: UV light, Radiation, Chemicals) DNA replication is error prone – base pair mismatches are sometimes introduced by DNA Polymerase Lack of correction can lead to mutations, a risk factor for cancer Consequences of DNA Mismatch Repair Deficiency Accumulation of mutations in DNA can cause cancer. Loss of mismatch repair (MMR) is linked to significantly increased cancer risk in a condition known as Lynch Syndrome. This disorder is characterized by a predisposition to early onset of colorectal and other internal cancers. Knowing who is at risk for cancer because of MMR deficiency can influence treatment options for existing patients or surveillance strategies for higher risk people. DNA Mismatch Repair (MMR) Evolutionarily conserved process Mechanism involves several protein dimers necessary for successful repair MutLα is composed of two separate proteins, MLH1 and PMS2 MMR acts to maintain genomic integrity by correcting DNA replication errors Mechanism of Mismatch Repair In-Vitro strand choice is determined by nick in non-template strand G Recognition MutS T MutL Exonuclease Excision T A DNA polymerase Resynthesis T Project Goals Goals: Establish experimental conditions for an in-vitro repair assay Compare four MLH1 mutants identified in human cancers vs. wild type MLH1 to see how/if the mutations affect MMR These mutants are: K751R, R755W, L582V, L607H [original amino acid] [location of mutation] [new amino acid] Approach: In-vitro repair assay with MP1 extract (MutLα deficient) plus recombinant MutLα Questions: What level of DNA repair activity will the mutants exhibit compared to the wild type? Amount of substrate repaired or kinetics of repair, for example Experimental Approach A plasmid containing a single known mismatch will be the substrate for the repair assays Recognition and correction of mismatch will restore the Xho1 restriction site on the plasmid MMR activity assessed by percentage of plasmid substrate that can be restricted Resynthesis Excision Ban1 restriction site Ban1 restriction site Restriction Ban1 restriction site 0 50 100 % repair Creation of Mismatch Containing Plasmid Substrate Start with the pRO1 plasmid pRO1 Nick plasmid Generate gap by adding excess of complementary oligonucleotide Anneal a DNA fragment of known sequence that will create a mismatch Mismatch substrate Experimental Approach – MP1 titration Question: How much non-MutL containing cell extract is saturating? MP1 cellular extract contains all MMR factors that are required for repair except MutLα A titration was done to identify conditions under which these factors aren’t limiting for repair Repair efficiency CT loop 80.00 60.00 % repair 0 70.00 No MutL 50.00 40.00 pk height 30.00 20.00 10.00 Increasing amounts of MP1 added 0.00 0 50 100 150 200 250 300 350 ug mp1 Repair is MutL, MP1 dependent Repair saturated around 225-250 µg Experimental Approach – MLH1 WT Kinetics Question: How does repair progress with time? MLH1 WT Kinetics 0 5 Minutes 10 15 20 25 30 35 40 45 50 55 60 80 70 % repair 60 50 40 Time points 30 20 10 Identical repair reactions stopped at different times 0 0 10 20 30 40 time (min) After 25 minutes no additional repair was observed 50 60 70 Results – MLH1 Mutant Endpoint Assay Question: Given a saturating amount of time, what levels of repair will the mutants exhibit? 2200 bp 1150 bp 1050 bp L607H L582V K571R R755W WT No MutL R755W appears to exhibit no repair at all L607H, L582V, K751R seem to repair about the same as WT Results – MLH1 Mutant Endpoint Assay continued.. Mutant Endpoint Repair Negative repair results from subtraction of negative control 70 60 50 (unidentified repair pathway) % repair 40 30 20 10 0 -10 -20 Construct L607H L562V K751R R755W WT Summary -MutLα is required for repair -225-250 µg MP1 extract is saturating -Repair saturates around 25 minutes -MutLα can be overexpressed in an active form -Activity can be reliably assayed -Kinetics are commensurate with previously published data -L607H, L582V, K751R appear to be capable of wild type levels of repair -R755W appears to exhibit no activity Future Studies -Refining the assay and protein quantitation -Expand substrate (CT, AG, AC, 8-oxo G, T dimers) -Different mutants Thanks to: Dr. Andrew Buermeyer Dr. Scott Nelson Dr. Kevin Ahern Howard Hughes Medical Institute (HHMI) Undergraduate Research, Innovation, Scholarship & Creativity (URISC)